Inhibition studies of metalloproteins by means of electrochemistry and spectroscopy Anton Nikolaev
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Anton Nikolaev. Inhibition studies of metalloproteins by means of electrochemistry and spectroscopy. Other. Université de Strasbourg, 2018. English. NNT : 2018STRAF053. tel-02146028
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ÉCOLE DOCTORALE DES SCIENCES CHIMIQUE UMR7140, Chimie de la matière complexe
THÈSE présentée par : Anton Nikolaev soutenue le : 29 octobre 2018
pour obtenir le grade de : Docteur de l’université de Strasbourg Discipline/ Spécialité : Chimie
Etudes d’inhibition de métalloprotéines par électrochimie et spectroscopie Inhibition studies of metalloproteins by means of electrochemistry and spectroscopy
THÈSE dirigée par : Pr HELLWIG Petra, Université de Strasbourg, Strasbourg, France RAPPORTEURS : Pr MIOMANDRE Fabien, Ecole normale supérieure de Cachan, Paris, France Dr LOJOU Elisabeth, BIP CNRS AMU, Marseille, France ______AUTRES MEMBRES DU JURY : Dr KIEFFER Bruno, Université de Strasbourg, Strasbourg, France
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Content Content ...... 2 Acknowledgments ...... 6 Abbreviations and acronyms ...... 7 Resumé en francais...... 9 Préface ...... 9 Partie A. Cytochrome bd-oxydase ...... 9 Partie B. La protéine mitoNEET ...... 16 List of the communications ...... 18 List of the publications ...... 18 Preface ...... 19 Part A. Cytochrome bd-oxidase ...... 20 Introduction ...... 20 I. The place of complex IV in the respiratory chain ...... 21 1. Bioenergetic membranes ...... 21 2. Components of respiratory chain ...... 22 Eukaryotic respiratory chain ...... 22 Cytochrome proteins and heme cofactors ...... 24 Prokaryotic respiratory chains ...... 25 ROS ...... 25 Quinols ...... 26 3. Cytochrome bd is a prokaryotic complex IV ...... 27 Classification of complex IV ...... 28 Structure of complex IV...... 29 A-type oxidases ...... 29 B- and C-type oxidases, NORs ...... 31 bd-oxidases ...... 32 Catalytic cycle ...... 35
aa3-oxidase ...... 35 bd-oxidase ...... 37 Function ...... 38 II. Inhibitors: role in the protein characterisation ...... 39 1. Kinetic parameters of enzyme catalysis ...... 39 2. Influence of the inhibitors on enzyme kinetics Inhibitor classification ...... 41 3. Inhibition measurements ...... 43 III. bd-oxidase and inhibitors ...... 45 IV. Development of electrochemical biosensor for the detection of inhibitors ...... 49 1. Biosensors ...... 49
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2. Protein film voltammetry ...... 50 Practical aspects ...... 50 Choice of the surface modification ...... 50 Gold electrode modified with GNPs covered with SAM ...... 54 Comparison of immobilised and solubilised complex IV ...... 54 The aims of the project ...... 56 Results ...... 57 I. Optimisation of the cyt bd immobilisation...... 58 1. Influence of concentration ...... 58 2. Stability of the signal ...... 59 Influence of the lipids ...... 60 Desorption or loss of the activity? ...... 61 Mutual influence of the surface charge of the SAM and lipid type ...... 61 Influence of the thiol spacer length ...... 63 Detergent variations ...... 64 3. Improvement of the preparation procedure ...... 65 GNPs deposition ...... 65 Thiol incubation ...... 67 The protein incubation at the surface ...... 69 4. Control experiments with HQNO ...... 69 5. Conclusions...... 71 II. The compound examination...... 72 1. Aurachin D derivatives examination...... 72 2. Various compound sets ...... 74 3. Conclusions...... 81 III. Cytochrome bd from G. thermodenitrificans ...... 82 1. Comparison with E. coli at pH7 ...... 82 UV/Vis-spectroscopy ...... 82 Spectroelectrochemistry ...... 83 UV/Vis titration ...... 83 Redox-induced differential FTIR spectroscopy ...... 87 Resonance Raman spectroscopy ...... 90 Electrochemistry ...... 92 2. The influence of the heme d on cyt bd properties...... 95 3. Dependency on pH and T ...... 96 Influence of T ...... 96 Influence of pH ...... 97 Mutual influence of pH and T ...... 98 4. Preliminary inhibition experiments ...... 101 5. Conclusions...... 103
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Part B. mitoNEET ...... 104 Introduction ...... 104 I. Structure ...... 105 II. Biophysical properties and mechanism ...... 106 III. Biological role ...... 107 IV. Influence of addition of compounds ...... 107 Aim of the PhD project ...... 108 Results and discussion...... 109 I. Prelimenary studies ...... 110 1. Cyclic voltammetry ...... 110 2. Resonance Raman spectroscopy ...... 111 3. Redox-induced differential FTIR spectroscopy ...... 113 II. Influence of phosphate ions...... 114 1. Cyclic voltammetry ...... 114 2. Resonance Raman spectroscopy ...... 115 III. Influence of pH ...... 115 1. Resonance Rama spectroscopy ...... 115 2. Redox-induced differential FTIR spectroscopy ...... 116 IV. Influence of the ligands ...... 118 1. Cyclic voltammetry ...... 118 2. Resonance Raman spectroscopy ...... 119 3. Redox-induced differential FTIR spectroscopy ...... 120 Conclusions ...... 121 General conclusions and perspectives ...... 122 Experimental part ...... 123 Methods ...... 124 I. Electrochemistry ...... 124 1. Cyclic voltammetry of adsorbed species ...... 124 2. Stationary electrochemistry ...... 126 II. Spectroscopy ...... 128 1. Absorption spectroscopy ...... 128 UV/Vis spectroscopy and its application towards proteins and cofactors...... 130 Mid-infrared spectroscopy of proteins ...... 132 2. Raman spectroscopy. Application for the study of protein cofactors ...... 135 Instrumentation ...... 139 I. Electrochemical equipment ...... 139 II. UV/Vis spectrometer ...... 139 Thin-layer electrochemical cell ...... 140 III. FTIR spectrometer ...... 141 Transmission cell ...... 142
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IV. Raman spectrometer ...... 143 V. Clark-electrode ...... 144 Experimental protocols ...... 145 I. Protein preparations ...... 145 1. Purification ...... 145 bd-oxidase from E. coli ...... 145 bd-oxidase from G. thermodenitrificans ...... 145 mitoNEET ...... 145 2. Pretreatment of the samples...... 145 PFV ...... 145 Differential FTIR spectroscopy ...... 147 Raman spectroscopy ...... 147 Electrochemical titrations ...... 147 FTIR transmission measurements ...... 147 Catalytic activity measurements in solution ...... 147 II. Electrode pretreatment and modification ...... 147 III. FTIR parameters ...... 148 IV. Raman spectroscopy measurements ...... 149 V. Redox titration experiments ...... 149 VI. IR transmission ...... 149 VII. Catalytic activity in solution ...... 150 Materials ...... 151 I. Compounds ...... 151 II. Gold nanoparticles preparation and validation ...... 151 III. Gold grid modification ...... 151 IV. Mediators ...... 152 V. Inhibitor addition ...... 153 Software ...... 153 Annexes ...... 154 References ...... 163 List of figures ...... 179 List of tables ...... 183 List of annexes ...... 185
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Acknowledgments
I would like to express my endless thanks to my supervisors Pr. Hellwig and Dr. Melin for their kind attitude regardless my performance in the laboratory. In particular, I would like to express my gratitude to Pr. Hellwig for granting me the opportunity to enter electrochemistry even if my background did not correspond at that time, and for encouraging in the time of frustrating experiments. It was a pleasure to discuss the ongoing experimental issues and result interpretations with Dr. Melin. Also I would like to thank Pr. Friedich for receiving me in his laboratory in Freiburg and Alex Thesseling for his excellent protein purifications.
I would like to make my acknowledgments to all collaborators contributing to my PhD project and members of the thesis committee accepting the proposition to be in the jury and dedicate their time for this.
I am grateful to our engineer Dr. Boubegtiten for her not only scientific but personal help and care. Especially, I will never forget that you did not leave me alone face-to-face with bureaucratic system. Due to this I also would like to make an acknowledgement to Ms Hnini with whom it was warm to speak every time. At the same time, I would like express my appreciation to Dr. El Khoury.
All PhD and master students I met in the laboratory were not less important for me. I will remember hilarious discussions with Sinan Sabunku and Natalia Grytsyk, Filipa Santos who could rescue almost every minute even if you was not in need, and Katherine Mezic who endured my linguistic jokes in English with comprehension.
I also would like to thanks my friends Guli Trukhmanova, Paul Jamme, Aleksandra Kostuk who spent their immense quantity of time for and with me. Special thanks I would address to Kathia Chernova carrying a piece of home with her and maintaining adventurous spirit in us.
I am grateful to many other of my acquaintances, to my teachers and to my alma mater Saint- Petersburg State University.
And the last but not the least, they are my dear parents Marina and Igor Nikolaevyi who are happy about my presence in spite of everything.
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Abbreviations and acronyms Adx ─ adrenoxine AOXs ─ alternative oxidase ATP ─ adenosine triphosphate BNC ─ binuclear centre CcO ─ cyt c oxidase CE ─ counter electrode CIO ─ cyanide-insensitive oxidase CNSs ─ carbon nanospheres CV ─ cyclic voltammetry cyt ─ cytochrome DDM ─ n-Dodecyl β-D-maltoside DET ─ direct electron transfer (DM)PE ─ 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine DMQ ─ 2-polyprenyl-3-methyl-6-methoxy-1,4-benzoquinone DMSO ─ dimethyl sulfoxide (DO)PG ─ 1,2-dioleoyl-sn-glycero-3-phosphoglycerol FAD ─ flavin adenine dinucleotide FAR ─ far infrared Fdx ─ ferredoxin FMN ─ flavin mononucleotide FTIR ─ Fourier-transform infrared spectroscopy GCE ─ glassy carbon electrode GNPs ─ gold nanoparticles Grx ─ glutaredoxin HCOs ─ heme-copper oxidases HQNO ─ 2-heptyl-4-hydroxyquinoline N-oxide HT ─ hexanthiol-1 HTS ─ high-throughput screening
IC50 ─ half-inhibition concentration IMAC ─ immobilized metal affinity chromatography IMM ─ inner mitochondrial membrane IMS ─ intermembrane space IC ─ internal conversion IR ─ infrared IRP-1 ─ iron regulatory protein 1 MCH ─ 6-mercapto-1-hexanol MCT ─ mercury cadmium telluride MEA ─ mercaptoethylamine MHA ─ 6-mercaptohexanoic acid MIR ─ middle IR MLCT ─ metall-ligand charge transfer
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MNG ─ maltose-neopentyl glycol mNT ─ mitoNEET MO ─ molecular orbital MOPS ─ 3-(N-morpholino)propanesulfonic acid MPA ─ 3-mercaptopropionic acid MQ or MK ─ menaquinone MUA ─ 11-mercaptoundecanoic acid MWCNTs ─ multi wall carbon nanotubes NAD(P)H ─ nicotinamide adenine dinucleotide (phosphate) NDH ─ NADH dehydrogenase NIR ─ near infrared NOR ─ NO reductase PCET ─ proton coupled electron transfer PFV ─ protein film voltammetry PGE ─ pyrolytic graphite electrode pio ─ pioglitazone pmf ─ proton motive force PQ ─ plastoquinol RDE ─ rotating disk electrode RE ─ reference electrode RHE ─ reversible hydrogen electrode RNS ─ reactive nitrogen species ROS ─ reactive oxygen species RR ─ resonance Raman rsv3S ─ resviratrol-3-sulfate SAM ─ self-assembled monolayer SHE ─ standard hydrogen electrode SQR ─ succinate-ubiquinone reductase SWCNTs ─ single wall carbon nanotubes tBLM ─ tethered bilayer membrane TMPD ─ tetramethyl-p-phenylenediamine TZD ─ thiazolidinedione UHDBT ─ undecylhydroxydioxobenzothiazole UQ ─ ubiquinone UV/Vis ─ ultraviolet/visible VR ─ vibrational relaxation WE ─ working electrode For the list of amino acid and their pK and pI values see Annex 1. Values of the physical constants mentioned in the thesis: • R=8.31 – universal gas constant, J mol−1 K−1 ⋅ ⋅ • F= 96485.33 – Faraday constant, C mol−1 ⋅ • c=3 108 – light velocity in vacuum, m -1 ⋅ ⋅s • h= 6.63 10-34 – Planck constant, m2 kg s-1 ⋅ ⋅ ⋅ 8
Resumé en francais
Préface
Les études d'interaction protéine-ligand permettent d'élucider la structure, le mécanisme catalytique des protéines et d'identifier les inhibiteurs. Les méthodes spectroscopiques et électrochimiques sont très efficaces pour la caractérisation des paramètres structuraux et fonctionnels des protéines et du complexe protéine-ligand1.
Dans notre travail, nous avons étudié deux types de métalloprotéines : l'hème contenant le cytochrome bd-oxydase et le FeS-cluster contenant la mitoNEET.
Partie A. Cytochrome bd-oxydase
Les mécanismes de conversion de l'énergie dans la cellule biologiques sont basés sur le gradient de protons électrochimique et aussi appelés la force motrice du proton, pmf. Le groupe des protéines donnant à la cellule la capacité de former un gradient essentiel est appelé chaîne respiratoire et il est localisé dans la membrane mitochondriale interne (dans le cas des eucaryotes) ou dans la membrane cellulaire (dans le cas des procaryotes). Toutes ces protéines, ou complexes, sont des métalloprotéines capables de transférer des électrons. Les électrons viennent des agents réduits (NADH, succinate) à travers la chaîne respiratoire des protéines, et au niveau du complexe IV sont ensuite transférés à l'accepteur terminal oxydatif qui est habituellement l'oxygène2. Le complexe IV a été classé en 3 familles: l'oxydase hème- cuivre, les bd-oxydases et les oxydases alternatives3. Dans nos études, nous nous sommes concentrés sur les bd-oxydases de type I exprimées chez Escherichia coli et chez le thermophile Geobacillus thermodenitrificans. Les bd-oxydases sont exprimées dans la cellule procaryote seulement et exclusivement dans des conditions microaérobiques sous lesquelles les agents pathogènes virulents peuvent se développer comme E. coli, M. tuberculosis, L. monocytogenes, K. pneumonia4. Une telle spécificité et une telle importance bioénergétique font du cytochrome bd une cible potentielle de nouveaux antibiotiques.
L'enzyme oxyde une molécule de quinol (UQ et MQ pour E. coli, MQ pour G. thermodenitficans) et réduit l'oxygène en eau. Au cours d'un cycle catalytique, deux protons sont libérés dans périplasme, dans le cas de bactéries Gram-négatives en formant le gradient de protons. La réaction totale est la suivante :